CD4+ helper T cells specific for human immunodeficiency virus type 1 (HIV-1) are associated with control of viremia. Nevertheless, vaccines have not been effective thus far, at least partly because sequence variability and other structural features of the HIV envelope glycoprotein deflect the immune response. Previous studies indicate that CD4+ T-cell epitope dominance is controlled by antigen three-dimensional structure. Three disulfide bonds in the outer domain of gp120 were individually deleted in order to destabilize the three-dimensional structure and enhance the presentation of weakly immunogenic epitopes. Unexpectedly, upon immunization of mice, the CD4+ T-cell response was broadly reduced and antibody titers were sharply increased for two of the disulfide variants. For one variant (deletion of the 296-331 disulfide bracketing V3), viral neutralizing activity was increased, but reactivity was narrow. For another variant (deletion of the 378-445 disulfide bracketing V4 and part of the bridging sheet), the antibody exhibited significant CD4-blocking activity. The changes in the immune response are most likely due to shifts in the pathways of antigen processing that result in the priming of fewer but more helpful T cells. In the proposed research, the disulfide variants will be reconstructed in the gp120 of distinct Clade B and Clade C HIV strains and in the gp120 of an SIV strain in order to test the generality of the result. Disulfide variants will be characterized by binding to monoclonal antibodies, circular dichroism spectroscopy with denaturation, limited proteolysis, deglycosylation, and isothermal titration calorimetry of CD4 binding. Mice will be immunized with the variants. CD4+ T-cell proliferative and cytokine responses will be mapped for individual mice and, in a novel analysis, will be correlated with antibody reactivity to proteins and peptides. The resulting epitope-specific T-B correlations will be used to identify cellular interactions that support antibodies directed against protective and unprotective epitopes. Rabbits will be immunized, and viral neutralization will be analyzed, with the expectation that antisera raised by the disulfide-deletion variants will have increased viral neutralization. The proposed research is unique in that it exploits T-B relationships in order to engineer an improved antibody response. PUBLIC HEALTH RELEVANCE: This project will test new strategies in vaccine engineering that may overcome the ability of the AIDS virus to resist immunity. Natural and engineered vaccine proteins will be used to immunize mice and rabbits, and then their immune responses will be analyzed in novel ways. We predict that the engineered vaccine protein will induce stronger immune responses against features of the virus that are not variable, and thus the immunity will be more effective against variants of the virus.